Figure 1.
Hepatic parasite burden and liver weight in C57BL/6 WT and Jα18-/- mice after infection with Leishmania donovani.
(A) Liver parasite burden determined on day 15, day 30, and day 60 post-infection by microscopic counting of Giemsa-stained tissue sections, and expressed as LDU (number of parasites/1000 nuclei × liver weight (mg)). (B) Change in liver weight over the course of infection in WT and Jα18-/- mice. Pooled data from two independent experiments (8–10 mice per group).
Figure 2.
Jα18-/- mice display a qualitatively and quantitatively impaired hepatic granulomatous response after infection with L.donovani.
Quantification of granuloma formation in the liver of L. donovani infected WT and Jα18-/-mice by microscopic examination of tissue sections stained with HES, at D15, D30 and D60 post-infection. Representative granuloma foci from WT mice at D15 (A) and D60 (B), and from Jα18-/- mice at D15 (C) and D60 (D) (×100 magnification). (E) Total number of granulomas in 100 microscopic fields (×400) at various time points. (F) Relative percentage of large granulomas (>25 cells) detected in each group of mice. (G) Relative percentage of mature granulomas detected in each group of mice.
Figure 3.
iNKT cells are recruited in the liver of WT mice on D15 after infection with L.donovani.
Hepatic cell infiltration of C57BL/6 WT and Jα18-/- mice was analyzed by flow cytometry 15 days after infection with L. donovani, using an anti-Gr1-FITC, anti-CD11b-PE-Cy7, anti-CD11c-APC, anti-CD4-PB, anti-CD3-FITC, anti-NK1.1-PerCP-Cy-5.5, and anti-CD8-APC-Cy7, and αGalCer-tetramer-PE. (A) Absolute quantification of hepatic NK (NK1.1+/CD3-), NKT (NK1.1+/CD3+), and TL (NK1.1-/CD3+) cell subsets in lymphocyte gated cells.(B) proportion of iNKT and non iNKT cell subsets in each group of mice in CD3+/NK1.1+ gated cells. (C) Absolute quantification of CD4+ and CD8+ cell subsets in gated NKT cells (CD3+/NK1.1+). (D) Absolute quantification of CD4+ and CD8+ cells in gated T lymphocytes (CD3+/NK1.1-). 106 cells of liver homogenates were labeled and data were analyzed on 50.000 events. Data are the mean ± SEM of four mice per group.
Figure 4.
Phenotypic characterization of the iNKT cell infiltrate in the liver of WT mice on day 15 after infection with L.donovani.
Hepatic cell infiltration of C57BL/6 WT was analyzed by flow cytometry using anti-CD3-V500, anti-NK1.1-PerCP-Cy-5.5, anti-TCRβ-V450, anti TCR γδ-FITC, and αGalCer/CD1d tetramer-APC, anti-CD4-PE-Cy7, anti-CD8-APC-Cy7. (A) Gating strategy allowing to estimate the low percentage of NK1.1- and NK1.1+ cells among TCRβ+/αGalCer/CD1d tetramer+ cells. (B) Gating strategy and evaluation of the percentage of γδ-T cells and non iNKT cells among CD3+/NK1.1+. (C) Percentage of CD4+ and CD4-/CD8- cells among iNKT cells (TCRβ+/αGalCer/CD1d tetramer+). This panel is representative of three independent experiments.
Figure 5.
Hepatic transcriptome of cytokines and receptors is altered in Jα18-/- mice.
Graphs represent the ratio of genes induction in Jα18-/- mice compared to WT, after normalization on three housekeeping genes, at day 0 (non infected) (A), day 15 (B), day 30 (C) or day 60 (D) after infection, with the central 45° angle line indicating a same level of induction in both backgrounds of mice and the two border lines corresponding to a 2-fold induction in one or the other group of mice. Data are the mean±SEM of triplicate samples pooled from 8 to 10 mice from two independent experiments. Quantification of mRNA of the T cell-chemoattractant chemokines CCL19 (E) and CXCL16 (F) in liver extracts of naïve WT and Jα18-/- mice, 15 days after infection. Data represent mRNA induction normalized on three housekeeping genes, and are the mean±SEM for each group of mice (8–10 mice per group from two independent experiments).
Figure 6.
Kinetics of hepatic mRNA induction and expression of key cytokines in WT and Jα18-/- mice infected with L.donovani.
Quantification of mRNA induction of IFN-γ (A), TNF-α (B), and IL-12 (C). Quantitative PCR was performed in liver extracts at various time points after infection and normalized by comparison to 18S mRNA. (D) Detection of TNF-α by ELISA in liver extracts. (E) Quantification of mRNA induction of IL-4. (F) Detection of IL-4 by ELISA in liver extracts. Data are the mean±SEM for each group of mice (8–10 mice per group from two independent experiments).
Table 1.
Spearman’s correlation rank between variables.
Figure 7.
Kinetics of selected chemokine and related chemokine receptors expression in liver extracts from WT and Jα18-/- mice.
mRNA expression of selected chemokines and their receptors was quantified by qPCR in liver extracts at various time points after infection. Quantification of mRNA for the T and NK cell-chemoattractant chemokines CXCL9, CXCL10 and their receptor CXCR3 (A), quantification of the PMN-chemoattractant chemokines MIP-2 and CXCL5 and their receptor CXCR2 (B) and of CCL2 and its receptor CCR2 (C). Data are the mean±SEM of 8 to 10 mice per group from two independent experiments and normalized on the expression of three housekeeping genes.
Figure 8.
Quantification of myeloid cells in the liver of wild-type and Jα18-/- mice.
(A) Representative slides of small (Day 15) and large (Day 60) hepatic granulomas labeled by immunohistochemistry using an anti-MPO antibody, in WT and Jα18-/- mice. (B) Proportion of MPO+ cells in granulomas, according to the size of the foci (i.e. small granulomas of <25 cells, and large granulomas >25 cells). (C) Quantification of the absolute number of MPO+ cells in granulomas in 100 consecutive microscopic fields (×400) at various time points. Data are the mean ± SEM for each group of mice (8–10 mice per group from two independent experiments). (D) Absolute quantification of myeloid cell infiltration in the liver of C57BL/6 WT and Jα18-/- by flow cytometry on D60 after infection with L. donovani, using an anti-Gr1-FITC, anti-CD11b-PE-Cy7, and anti-F4/80-PE. Analysis was done on live cells after excluding lymphocyte cells. 106 cells of liver homogenates were labeled and data were analyzed on 50.000 events. Data are the mean ± SEM of four mice per group. (E) Quantification of MPO mRNA expression in the liver of WT and Jα18-/- mice. Data are the mean±SEM for each group of mice (8–10 mice per group from two independent experiments).
Figure 9.
Hepatic lymphoid cell infiltration in wild-type and Jα18-/- mice at day 60 of infection.
Quantification of lymphoid cells in the liver of C57BL/6 WT and Jα18 by flow cytometry after infection with L. donovani, using anti-CD4-PE, anti-CD3-Pacific Blue, anti-NK1.1-PerCP-Cy-5.5, and anti-CD8-APC-Cy7. 106 cells of liver homogenates were labeled and data were analyzed on 50.000 events. Absolute quantification of hepatic NK (NK1.1+/CD3-), NKT (NK1.1+/CD3+), and TL (NK1.1-/CD3+) cell subsets (A), absolute quantification of CD4+ and CD8+ cell subsets in gated TL (B). Data are the mean ± SEM of four mice per group.